Detection of DNA in specimens comprising body fluids of tissues can be difficult because of the small quantity of DNA present or because of the presence in the specimen of other interfering materials, including DNA from a different source. These limitations may be overcome by employing an analytic method referred to as the polymerase chain reaction (PCR) technique. By this technique, selective enrichment of a specific DNA sequence can be achieved by exponential amplification of the target sequence. Mullis, et al., Meth Enzymol., 155, 335 (1987).
To facilitate PCR amplification, pairs of oligonucleotide primers may be employed as described in U.S. Pat. No. 4,683,202 (hereby incorporated by reference). The primers are designated to hybridize with sequences that flank the target DNA. Following in vitro amplification, the amplified target sequence is detected by a hybridizing probe. For example this analytical procedure has been used for the direct detection of HIV-1 as described by Ou, et al., Science,238:295-97 (1988). The amplification cycles are facilitated by using a polymerase which is thermally stable in incubations up to 95.degree. C. as described by Saiki, et al., Science, 239:487-91 (1988).
E. suis is an extracellular red blood cell parasite that causes icteroanemia in acutely ill pigs and a variety of syndromes in chronically infected pigs. Current techniques to detect E. suis infection are limited by the variability of parasitemia and the antibody response in the infected animal. In particular, diagnosis of eperythrozoonosis is complicated by lack of readily identifiable parasitemia in latent and chronic infections, rapid loss of parasitemia at the time of onset of clinical signs in acutely ill pigs and the lack of sensitive and specific laboratory tests for the organism.
Serological tests for eperythrozoonosis include an indirect hemagglutination test (IHA), Smith et al., J. Am. Vet. Med. Assoc., 36:1319-20 (1975) and an enzyme linked immunosorbent assay (ELISA), Hsu et al., J. Am. Vet. Med. Assoc.., 53:352-54 (1992). The ELISA test provided superior results to the IHA test in sensitivity; but, both of these tests share limitations because of the nature of the antibody response to E. suis in pigs. Young pigs, i.e. less than twelve weeks, and boars have low titers to the organism and the antibody titers tend to decline rapidly in chronically infected individuals. Because of this variability in the antibody response, the standard ELISA and IHA tests are more useful as tests to screen herds for E. suis but have limited value in diagnosis of acute eperythrozoonosis.
Genetic approaches to diagnosing eperythrozoonosis have been reported. Oberst, et al., Am. Vet. Res., 51:1760-64 (1990 hereby incorporated by reference). A recombinant DNA probe to E. suis was used to detect E. suis DNA extracted from the blood of experimentally infected, splenectomized pigs. The DNA probe (KSU-2) was able to detect infection seven days post infection, which corresponds with the appearance of clinical signs and detectable parasitemia on blood smears. Although this probe is specific for detecting E. suis in acutely ill pigs, it was unable to detect infections during the prepatent period (i.e. days 1 through 6) before the pigs become clinically ill. As such a need existed for an efficient diagnostic test which can be used to detect early infection with E. suis so that steps can be taken prior to the outbreak of clinical symptoms.